人羊水来源干细胞的实验研究

发布时间：2018-05-31 10:04

本文选题：羊水 + 干细胞　；参考：《吉林大学》2012年博士论文

【摘要】：糖尿病是由多种病因引起的以慢性高血糖为特征的代谢性疾病，严重危害着人类健康。目前胰岛素注射是临床上治疗糖尿病的主要方法，可以短期内控制血糖。不足之处是需要频繁监测血糖及胰岛素注射，而且不能阻止糖尿病所引起的肾衰、心脏病变、眼底病变、坏疽等并发症。近年来胰腺移植和胰岛细胞移植治疗糖尿病取得了很大进展，可有效改善患者内源性胰岛素的分泌，改善患者生活状态。胰岛移植最大的问题就是胰岛供体来源不足和终身使用免疫抑制剂。目前解决细胞移植供体不足最有可能的就是干细胞。利用干细胞体外诱导分化为胰岛素分泌细胞，不仅可以从根本上解决细胞来源问题，还可以克服免疫排斥问题。羊水中存在着多潜能干细胞。人羊水干细胞易于获取，可以从产前诊断的少量羊水中提取；也可以从产后的羊水中分离。而且抽取羊水不会损害母体，也不会对胚胎造成影响，也就不存在伦理和道德上的问题。羊水中的细胞是一群处于胚胎发育早期的细胞群。从中分离的人羊水干细胞(amniotic fluid-derived stemcells, hAFS)细胞是介于胚胎干细胞(embryonic stem cells, ES)和成体干细胞之间的细胞类型，表达ES细胞和成体干细胞的标志，具有相似的多向分化潜能，可以分化为三个胚层的细胞。hAFS细胞体外比较容易培养，其增殖较快，动物体内移植不会形成畸胎瘤，成为干细胞研究的热点。本实验从羊水中分离hAFS细胞，以hAFS细胞为研究对象。研究hAFS细胞的生物学特性，以及多向分化潜能，重点研究其分化为胰岛素分泌细胞的能力。建立及优化hAFS细胞诱导分化的实验方案，探讨诱导后细胞的功能和活性，为其临床应用提供依据。 1. hAFS细胞的分离培养和生物学特性从羊水中分离获得hAFS细胞。通过流式细胞仪和免疫荧光测定，hAFS细胞具有ES细胞和间充质干细胞(mesenchymal stem cells, MSCs)的标志，同时也首次检测到SSEA-1在hAFS细胞中的表达。hAFS在体外可以大量扩增，具有较强的增殖能力。通过细胞周期和端粒酶测定，也证实细胞具有典型干细胞的特点。超微结构发现细胞表面大量的微绒毛，胞内富含丰富的细胞器，，说明获得的hAFS细胞功能活跃，具有强蛋白合成的能力，维持自身的增殖和分化。 2. hAFS细胞的多向分化潜能本实验对hAFS向脂肪细胞、成骨细胞和神经细胞三个方向进行诱导分化。通过油红O染色、茜素红染色、Von Kossa’s染色、RT-PCR和免疫组化等实验证实了hAFS细胞在体外特定诱导条件下可以分化为脂肪细胞、成骨细胞和神经细胞。同时本实验也分析了hAFS细胞的致瘤性。将第3代，第5代，第8代细胞以5×10~6、1×10~7、2×10~7个细胞注射BALB/C裸鼠皮下，观察两个月，未发现肿瘤的形成。说明hAFS细胞动物体内移植不具有致瘤性，这也为临床应用提供可能。 3. hAFS细胞分化为胰岛素分泌细胞本实验以胰腺体内发育为依据，诱导hAFS细胞分化为胰岛素分泌细胞。首先bFGF和尼克酰胺联合诱导hAFS细胞分化为胰腺祖细胞。通过检测，这些细胞表达胰腺前体细胞相关的转录因子，例如Pdx-1、Nng3、Pax4。之后加入EGF和exendin-4联合诱导胰腺祖细胞分化为胰岛素分泌细胞。EGF可以有效促进Pdx-1阳性的胰腺祖细胞增殖，exendin-4可促进细胞内分泌方向分化。这些胰岛素分泌细胞表达胰岛相关的转录因子，如Pdx-1、Nkx6.1等，同时表达胰岛功能相关的功能基因，如胰岛素、葡萄糖转运因子和葡萄糖激酶等。最后对这些胰岛素分泌细胞进行葡萄糖刺激试验，发现这些细胞对葡萄糖刺激敏感，根据葡萄糖浓度释放相应的胰岛素。总之，本实验从羊水中分离得到一种新的hAFS细胞，hAFS细胞具有较强的增殖能力和多向分化潜能，体内移植不具有致瘤性。同时高效诱导hAFS细胞分化为胰岛素分泌细胞，这些细胞具有类似成人胰岛的功能和体内发育模式。这为糖尿病临床治疗提供重要的理论和实验基础。
[Abstract]:Diabetes is a metabolic disease characterized by a variety of causes of chronic hyperglycemia, which seriously endangers human health. At present, insulin injection is the main method for clinical treatment of diabetes, which can be used to control blood glucose in the short term. The deficiency is that frequent monitoring of blood glucose and islet injection can not be used to prevent diabetes. Renal failure, heart disease, fundus lesion, gangrene and other complications. In recent years, pancreatic transplantation and islet cell transplantation have made great progress in the treatment of diabetes, which can effectively improve the secretion of endogenous insulin and improve the patient's living condition. The biggest problem of islet transplantation is the lack of islet donor and the life-long use of immunosuppressive agents. The most likely to solve the deficiency of cell transplantation donor is stem cells. The use of stem cells to induce differentiation into insulin secreting cells in vitro can not only solve the problem of cell origin fundamentally, but also overcome the problem of immune rejection.
There are pluripotent stem cells in amniotic fluid. Human amniotic fluid stem cells are easy to obtain, can be extracted from a small amount of amniotic fluid diagnosed by prenatal, and can be separated from postpartum amniotic fluid. And amniotic fluid extraction does not damage the mother body, does not affect the embryo, and there is no ethical and moral problems. The cells in the amniotic fluid are in a group. Amniotic fluid-derived stemcells (hAFS) cells are the cell types between embryonic stem cells (embryonic stem cells, ES) and adult stem cells, which represent the markers of ES and adult stem cells, which have similar multidirectional differentiation potential and can be divided into three. The.HAFS cells of the germ layer are easy to culture in vitro, and their proliferation is faster. The transplantation of animals will not form teratoma, which has become the focus of stem cell research.
In this experiment, the hAFS cells were isolated from amniotic fluid, and hAFS cells were used as the research object. The biological characteristics of hAFS cells and the potential of multidifferentiation were studied. The ability to differentiate into insulin secreting cells was studied. The experimental scheme of inducing and optimizing the induction of differentiation of hAFS cells was established and optimized to explore the function and activity of the induced cells to provide the clinical application. Basis.
Isolation, culture and biological characteristics of 1. hAFS cells
HAFS cells were isolated from amniotic fluid. By flow cytometry and immunofluorescence, hAFS cells had ES and mesenchymal stem cells, MSCs, and the expression of SSEA-1 in hAFS cells was also detected for the first time. The proliferation ability of.HAFS in vitro was stronger. The cell cycle was through cell cycle. The determination of telomerase and telomerase showed that the cells have the characteristics of typical stem cells. The ultrastructure found a large number of microvilli on the surface of the cells and rich organelles in the cell, indicating that the acquired hAFS cells have active function, strong protein synthesis ability, and maintain their own proliferation and differentiation.
Multidirectional differentiation potential of 2. hAFS cells
In this experiment, hAFS was induced to differentiate into adipocytes, osteoblasts and nerve cells in three directions. Through the oil red O staining, alizarin red staining, Von Kossa 's staining, RT-PCR and immunohistochemistry, it was proved that hAFS cells could be differentiated into adipocytes, osteoblasts and nerve cells under specific induction conditions in vitro.
At the same time, the tumorigenicity of hAFS cells was also analyzed. Third, fifth, and eighth generation cells were subcutaneously injected with 5 x 10~6,1 x 10~7,2 x 10~7 cells to BALB/C nude mice. It was observed for two months and no tumor formation was found. It showed that the transplantation of hAFS cells in vivo did not have the tumorigenicity, which also provided the possibility for clinical application.
3. hAFS cells differentiate into insulin secreting cells
This experiment, based on the development of the pancreas, induces hAFS cells to differentiate into insulin secreting cells. First, bFGF and nicotinamide are combined to induce hAFS cells to differentiate into pancreatic progenitor cells. Through detection, these cells express the transcription factors related to the precursor cells of the pancreas, such as Pdx-1, Nng3, and Pax4., and join the EGF and exendin-4 to induce the pancreas. The differentiation of progenitor cells into insulin secreting cells.EGF can effectively promote the proliferation of Pdx-1 positive pancreatic progenitor cells, and exendin-4 promotes the direction differentiation of cell endocrine cells. These insulin secreting cells express islet related transcription factors, such as Pdx-1, Nkx6.1, etc., and express the functional genes related to islet function, such as insulin, glucose transport Factors and glucokinase and so on. Finally, the glucose stimulation tests on these insulin secreting cells showed that these cells were sensitive to glucose stimulation and released the corresponding insulin according to the glucose concentration.
In conclusion, a new hAFS cell was isolated from amniotic fluid. HAFS cells have strong proliferation ability and multidirectional differentiation potential. In vivo transplantation does not have tumorigenicity. At the same time, hAFS cells are highly induced to differentiate into insulin secreting cells, which are similar to adult pancreatic islets and in vivo development patterns. Bed therapy provides an important theoretical and experimental basis.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

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